The study of transcription and replication of the Marburg virus using a minireplicon system constructed on the basis of viral genome [corrected].

Abstract

A minireplicon system based on the Marburg virus genome that does not require the infectious virus for studying its transcription and replication was constructed. This system comprises the support plasmids that encode full-length copies of four genes of Marburg virus nucleocapsid proteins under the control of T7 RNA polymerase and a minigenome-encoding plasmid. The minigenome construct consists of noncoding terminal regions of the Marburg virus genome and the reporter gene of chloramphenicol acetyltransferase. It has been shown that the noncoding regions contain the cis -acting transcription and translation regulatory elements that are recognized by both wild-type Marburg virus and the recombinant polymerase complex synthesized using the support plasmids. We discovered that the Marburg virus proteins VP35, NP, and L are contained in the polymerase complex that is involved in the minigenome transcription and replication, whereas the VP30 nucleocapsid protein is not an essential component of the polymerase complex. Transcription of mRNA and replication of the miniantigenome RNA were demonstrated with the use of Northern blot hybridization. Both minigenome and miniantigenome RNA are associated with the nucleocapsid proteins, which protects them from nucleases. Marburg and Ebola viruses belong to the Filoviridae family. They cause severe hemorrhagic fever in primates and humans with high lethality [1]. Currently, the researchers work on constructing the recombinant systems that allow direct genetic engineering manipulations with the genomes of these viruses so as to study the molecular bases of their extremely high pathogenicity and to develop treating and preventing procedures [2‐4]. The insertion of certain mutations into the viral genome using genetic engineering procedures allows studying the mutation-induced changes in the replication of viruses and their interaction with susceptible cells. This system of “reverse genetic” methods developed earlier for some viruses with negative RNA